Project Summary: Prion diseases are neurodegenerative disorders that pose a threat to human and environmental health. These illnesses have genetic and nongenetic causes, can be environmentally acquired, and have the ability to remain infectious in the absence of a host organism for extended periods. Aggregation of misfolded prion protein (PrPSc) correlates with disease, but the mechanism behind aggregation is not fully understood, gravely hindering the development of therapeutics to prevent fibrillation and sickness. The ?2?2 loop of mammalian prion protein has previously been demonstrated to be a key region implicated in disease transmission. A crystal structure from a nine-residue segment encoding the ?2?2 loop of the bank vole prion has demonstrated structural characteristics and stability characteristic of full-length prion fibers. Building on this structure, structures of PrP aggregates that convey information on stability and infectivity will be pursued. To achieve this goal, ordered aggregates of PrP segments that have been described to play a role in disease transmission in various species with a range of prion disease susceptibility will be biochemically and structurally characterized. Constructs incorporating disease modulating regions will be recombinantly expressed, purified, and fibrillized to assess stability, proteinase K resistance, and fiber morphologies. These properties will be compared against those observed in fibrils derived from diseased animals. A combination of crystallography and single particle Cryo-EM will be used to determine the atomic arrangement of each misfolded PrP. These aims will be achieved through the application of frontier methods in macromolecular crystallography including electron micro-diffraction (MicroED). The resulting structures will provide a molecular explanation for a nearly three- hundred-year-old mystery in prion biology and protein pathology and will help distinguish infectious from non- infectious amyloids, revealing structural code for prion transmission barriers. !